This invention relates to nucleic acid and amino acid sequences of human nucleic acid methylases and to the use of these sequences in the diagnosis, treatment, and prevention of cancer and developmental disorders.
Methylation of nucleic acids is a common regulatory mechanism used by all organisms. In prokaryotes, methylation prevents degradation of cellular DNA by restriction enzymes. In eukaryotes, methylation has been shown to play a role in regulating gene transcription as well as participating in the proof-reading step in DNA replication. The methylation of ribosomal RNA is necessary for the proper folding and function of ribosomes, which are essential in protein translation. Thus, nucleic acid methylation is involved in a number of essential cellular processes.
Ribosomal RNA (rRNA) is the most abundant type of RNA, comprising 80% of the ribonucleic acid in an E. coli cell. The precise function of rRNA remains unclear, but it is known that rRNA is the nucleic acid constituent of ribosomes, which are essential components of the protein translation machinery. A number of chemically modified nucleic acids are included in rRNA, most of which are methylated or dimethylated versions of the standard RNA components. Specific RNA methylases transfer either one or two methyl groups from a donor, typically S-adenosyl methionine, to nitrogen atoms of the target nucleic acid. A second type of modification involves deamination of certain adenosine residues to form inosine, which may then be methylated. The modification of select nucleic acids in rRNA is critical to ribosomal function, and therefore to protein translation in general. For example, in certain bacteria, inactivation of the gene encoding RNA dimethylase interferes with bacterial resistance to antibiotics such as erythromycin. (Lafontaine, D. et al. (1994) J. Mol. Biol. 241:492-497.)
There are two classes of DNA methylases, differing in the nature of the modification produced. The first class methylates a ring carbon of cytosine, to produce C5-methylcytosine. The second class methylates exocyclic (non-ring) nitrogens in adenine or cytosine, to produce either N6-methyladenine or N4-methylcytosine. Both classes use S-adenosyl methionine as a methyl-group donor.
The methylation of DNA plays two distinct roles in eukaryotic organisms. One critical step during cell proliferation and growth is the accurate replication of DNA. Ensuring the fidelity of DNA replication is crucial to the survival of the organism. This is accomplished by the proof-reading function of DNA polymerases, such as the subunit of DNA polymerase III, which compares the newly synthesized strand against the template strand. The question of strand identity is answered by the presence of methylated adenosines. Since the modification of adenosine by DNA methylase does not occur until after replication, only the template strand will be methylated. In this way, DNA methylation serves as a chronological marker, to distinguish between new and old DNA strands.
DNA methylation also plays an important role in regulating gene transcription. In vertebrates, methylation prevents gene transcription by interfering with the binding of transcription factors, and active genes are typically not highly methylated. Neoplastic cells often demonstrate hypermethylation in certain regions of DNA, and increased DNA methylase activity. In particular, regions of DNA known as promoter-region CpG islands are targets for excess methylation. Extensive methylation of CpG islands prevents the activation of certain tumor-suppressor genes, such as the retinoblastoma gene (Rb), the von Hippel-Lindau gene (VHL), and E-cadherin. Preventing the activation of tumor suppressor genes results in loss of tumor suppression and promotes neoplastic transformation. In general, changes in global levels of methylation and regional changes in patterns of methylation (e.g., CpG islands), are among the earliest and most frequently observed events known in many human cancers. For this reason, the activity of DNA methylases can provide an early screen for cancer detection. In addition, recent evidence suggests that changes in methylation of DNA in promoter regions may be involved other, non-cancerous disease states. The atrial natriuretic peptide (ANP) system is implicated in the pathophysiology of primary open-angle glaucoma. Analysis of the 5xe2x80x2 proximal promoter region of the ANP gene revealed mutations that would lead to changes in the methylation state of the region. Thus, DNA methylation level may affect the transcription of ANP and contribute to the pathology in certain cases of glaucoma. (Graff, J. R. et al. (1997) J. Biol. Chem. 272:22322-22329; Baylin, S. B. et al. (1998) Adv. Cancer Res. 72:141-196; Gonzalgo, M. L. and Jones P. A. (1997) Mutat. Res. 3866:107-118; Tunny, T. J., et al. (1996) Biochem. Biophys. Res. Commun. 223:221-225.)
The discovery of new human nucleic acid methylases and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer and developmental disorders.
The invention features substantially purified polypeptides, human nucleic acid methylases, referred to collectively as xe2x80x9cHNAMxe2x80x9d and individually as xe2x80x9cHNAM-1xe2x80x9d and xe2x80x9cHNAM-2.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2, as well as a purified agonist and a purified antagonist to the polypeptide. The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2. The invention also provides a method for treating or preventing a developmental disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:2 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.